SPMYB FUNCTIONS AS AN INTRAMODULAR REPRESSOR TO REGULATE SPATIAL EXPRESSION OF CYIIIA IN SEA-URCHIN EMBRYOS

The CyIIIIa actin gene of Strongylocentrotus purpuratus is transcribed exclusively in the embryonic aboral ectoderm, under the control of 2. 3 kb cis-regulatory domain that contains a proximal module that contro ls expression in early embryogenesis, and a middle module that control s expression in later embryogenesis. Previous studies demonstrated tha t the SpRunt-l target site within the middle module is required for th e sharp increase in CyIIIa transcription which accompanies differentia tion of the aboral ectoderm, and that a negative regulatory region nea r the SpRunt-l target site is required to prevent ectopic transcriptio n in the oral ectoderm and skeletogenic mesenchyme, This negative regu latory region contains a consensus binding site for the myb family of transcription factors. In vitro DNA-binding experiments reveal that a protein in blastula-stage nuclei interacts specifically with the myb t arget site, Gene transfer experiments utilizing CyIIIa reporter constr ucts containing oligonucleotide substitutions indicate that this site is both necessary and sufficient to prevent ectopic expression of CyII Ia, Synthetic oligonucleotides containing the myb target site were use d to purify a protein from sea urchin embryo nuclear extracts by affin ity chromatography, This protein is immunoprecipitated by antibodies s pecific to the evolutionarily conserved myb domain, and amino acid seq uences obtained from the purified protein were found to be identical t o sequences within the myb domain. Sequence information was used to ob tain cDNA clones of SpMyb, the S, purpuratus member of the myb family of transcription factors, Through interactions within the middle modul e, SpMyb functions to repress activation of CyIIIa in the oral ectoder m and skeletogenic mesenchyme.